Large scale processes are known in the art and are necessary for the industrial production of various materials. Since large scale processes cannot be performed by the same means as small scale processes, specific processes for the large scale production of materials must be designed, even if small scale processes exist.
Nutraceuticals are sometimes prepared using synthetic processes that provide the desired active ingredients, e.g., polyphenols, which are naturally found in fruit cells. However, the use of synthetic processes does not provide the natural ingredients along with the active ingredients, which sometimes contribute to the efficiency of the formulation.
Other types of nutraceuticals are prepared from the natural plants; however, all known large scale processes for preparing nutraceuticals from plants include the extraction of the prepared plant cells in order to obtain the desired active ingredient. However, when plants containing polyphenols, for example, are extracted, the final product may be hitter. Also, only certain parts of the plant may be successfully extracted since only they contain the desired amounts of the active ingredients.
Small scale processes for the preparation of fruit cells are known in the art; however, large scale processes are more difficult to design since they tend to amplify the production of the primary metabolites, while minimizing the productions of the secondary metabolites. Since active ingredients, such as polyphenols, are secondary metabolites their production in large-scale processes is complex.
Nutraceuticals derived from polyphenol-containing fruit extracts are known for their beneficial effects. However, it has been shown that the therapeutic effect of fruit extracts is dependent on species, location, year (annual climate), processing etc. and therefore reliance on natural fruits as a source of these regulatory compounds does not lead to a homogeneous or consistent supply of material. Furthermore, fruits are often contaminated by residual fungicides, pathogens, pesticides and pollutants.
Nonetheless, dietary consumption of polyphenols was shown to be inversely related to morbidity and mortality from coronary heart disease (CHD). Moreover, an inverse association between polyphenols intake and subsequent occurrence of ischemic heart disease, or cerebrovascular disease was shown. Over the last decade, studies indicated that pomegranate is a potent antioxidant and its therapeutic properties further include treatment and prevention of cardiovascular disease, erectile dysfunction, dental conditions and protection from ultraviolet radiation. Other potential applications include infant brain ischemia, Alzheimer's disease, arthritis and obesity.
Thus, there is a need in the art for a large scale process for preparing fruit cells from natural ingredients, which includes the production of both the primary and the secondary metabolites of the fruit cells. There is need for natural (phyto) compositions that may be prepared in a large scale process in which the amount of the active ingredient is consistent and recurrent (e.g., clonal preparations), is highly bioavailable and easily administered for the treatment and prevention of various diseases and disorders.
Pomegranate juice and fruit extracts (including seeds, inner lamellae, mesocarp and exocarp), as well as plant parts, such as, bark, roots, and leaves, exhibit potent biological properties attributable to the presence of polyphenols. Polyphenols content of pomegranate includes flavonoids, phenolic acids and tannins, all of which are present in various plant parts, such as, bark, leaf and fruit seeds, inner lamellae, mesocarp and exocarp. Within the fruit, juice polyphenols include mainly anthocyanins such as cyanidin-3-glycoside, cyanidin-3,3-diglycoside, and delphindin-3-glucoside) and anthoxanthins (such as catechins, ellagic tannins, and gallic and ellagic acids, whereas hydrolysable ellagitannins are found mostly in the peels.
Ellagic acid and hydrolysable ellagitannins are both implicated in protection against atherogenesis, along with their potent antioxidant capacity. Punicalagin is the major ellagitannin in pomegranate, and this compound is responsible for the high antioxidant activity of this juice. An additional polyphenol found in pomegranate (mainly in the leaves) is 1,2,3,4,6-pentagalloyl glucose (PGG). PGG is a component of plants traditionally used in Chinese medicine, as well as in other fruits such as mango and banana. PGG has recently been shown to prevent biofilm formation by S. aureus and bind insulin receptor and thus to activate insulin-mediated glucose transport signaling pathway and to induce p53 and apoptosis in cancer cells through insulin receptor signaling.
Due to the extensive knowledge about the pomegranate's health attributes and increasing public awareness about functional food, the demand for pomegranate fruit and its byproduct has increased tremendously in the western world. As a result of this trend, the extent of pomegranate growth was increased significantly in many regions throughout the world, and industries that produced pomegranate products have been developed.
Pomegranate-derived callus cultures have been generated by several research groups. However, the cultures developed so far have been used for in vitro organogenesis and plant regeneration and the expression of secondary metabolites as an ingredient in nutraceutical products was not tested Therefore, there is a need in the for the production of a pomegranate cell culture-based product that includes both the primary and the secondary metabolites of the pomegranate cells.